Biomedical subjects

Catherine Souchier

Publications and source records attributed to Catherine Souchier.

4 recordsLinked to original sources

PubMed2005

Monitoring the interaction between DNA and a transcription factor (MEF2A) using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is an analytical method that allows distinguishing different populations of fluorescent probes in solution and provides data on their concentrations and their diffusion coefficients. FCS was used to characterize the interaction of the transcription factor (MEF2A) with its DNA target sequence. The myocyte enhancer factor 2 (MEF2) belongs to the MADS-box family and activates transcription of numerous muscle genes during myogenesis. Measurements were made using TAMRA-labelled oligonucleotide duplexes derived from a wild type (WT) or a mutated MEF2 target gene. Binding of the protein to the WT DNA resulted in significant changes of the diffusion. Specificity of the interaction was confirmed using the mutated DNA. Bound to free probe ratios were determined at different MEF2A concentrations and the apparent equilibrium dissociation constant K(D) for the full-length MEF2A was estimated.

Base Sequence↗

PubMed2005

Highlighting protein kinase CK2 movement in living cells.

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (alpha2beta2) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol 23: 975-987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio-temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.

3T3 Cells↗

PubMed2003

Data reproducibility in fluorescence image analysis.

Fluorescence image analysis provides quantitative data on fluorescence in situ hybridization signals (FISH), immunofluorescence labelings, Green Fluorescent Protein (GFP) expression and microarrays. It is a valuable tool for decision making in the fields of biology and medicine. The aim of this study was to evaluate the reproducibility of fluorescence intensity measurements and standardization when acquisitions are performed under various but well defined conditions. Fluorescent intensity of standard beads (Inspeck series, Molecular Probes) was repeatedly measured using an image analyzer and automated procedures. Images were acquired using several integration times and neutral filter sets. A standardization procedure was used for expressing the data in a same unit: data were multiplied by the light attenuation factor and were divided by the CCD integration times. Results show that 1) standardization is possible 2) accurate and reliable fluorescence measurements can be obtained and 3) specimens showing large differences in fluorescence intensity can be objectively compared. Moreover fluorescent test slides including fluorochrome solutions and altuglas slides were tested for shading correction and as overall test systems.

Calibration↗

PubMed2003

Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual subunits.

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (alpha or alpha') and two regulatory (beta) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2alpha or GFP-CK2beta revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2beta, CK2alpha can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2alpha is dramatically changed by its association with CK2beta, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.

3T3 Cells↗